5,7-disubstituted thiazolo[4,5-d]pyrimidines for the selective inhibition of chemokine receptors

ABSTRACT

There are disclosed novel 5,7-disubstituted [1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one derivatives of formula (I) 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3 , R 4  and n are as defined in the specification, and pharmaceutically acceptable salts thereof, together with processes for their preparation, pharmaceutical compositions comprising them and their use in therapy. The compounds of formula (I) are CX 3 CR1 receptor antagonists and are thereby particularly useful in the treatment or prophylaxis of neurodegenerative disorders, demyelinating disease, cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, rheumatoid arthritis, pulmonary diseases such as COPD, asthma or pain.

FIELD OF THE INVENTION

The present invention discloses novel 5,7-disubstituted[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one derivatives together withprocesses for their preparation, pharmaceutical formulations comprisingthem and their use in therapy.

BACKGROUND OF THE INVENTION

Chemokines play an important role in immune and inflammatory responsesin various diseases and disorders, including asthma, atherosclerosis andallergic diseases, as well as autoimmune pathologies such as rheumatoidarthritis and multiple sclerosis. These small, secreted molecules are agrowing superfamily of 8-14 kDa proteins characterised by a conservedcysteine motif. At the present time, the chemokine superfamily comprisesfour groups exhibiting characteristic structural motifs, the C—X—C, C—Cand C—X₃—C and XC families. The C—X—C and C—C families have sequencesimilarity and are distinguished from one another on the basis of asingle amino acid insertion between the NH-proximal pair of cysteineresidues. The C—X₃—C family is distinguished from the other two familieson the basis of having a triple amino acid insertion between theNH-proximal pair of cysteine residues. In contrast, members of the XCfamily lack one of the first two cysteine residues.

The C—X—C chemokines include several potent chemoattractants andactivators of neutrophils such as interleukin-8 (IL-8) andneutrophil-activating peptide 2 (NAP-2).

The C—C chemokines include potent chemoattractants of monocytes,lymphocytes and neutrophils. Examples include human monocyte chemotacticproteins 1-3 (MCP-1, MCP-2 and MCP-3), RANTES (Regulated on Activation,Normal T-cell-Expressed and Secreted), eotaxin and the macrophageinflammatory proteins 1α and 1β (MIP-1α and MIP-1β).

The C—X₃—C chemokine (also known as fractalkine) is a potentchemoattractant and activator of microglia in the central nervous system(CNS) as well as of monocytes, T cells, NK cells and mast cells.

Studies have demonstrated that the actions of the chemokines aremediated by subfamilies of G protein-coupled receptors, among which arethe receptors designated CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C—C family); CXCR1,CXCR2, CXCR3, CXCR4 and CXCR5 (for the C—X—C family) and CX₃CR1 for theC—X₃—C family. These receptors represent good targets for drugdevelopment since agents that modulate these receptors would be usefulin the treatment of disorders and diseases such as those mentionedabove.

WO 01/25242 discloses certain thiazolo[4,5-d]pyrimidine derivatives thatare useful as antagonists of receptors linked to the C—X—C and C—Cchemokine families, particularly as antagonists of the CXCR2 receptor.

The present invention relates to a group of compounds that are relatedto compounds disclosed in WO 01/25242 but are of a structural type notspecifically exemplified therein. When compared to the Examplesdisclosed in WO 01/58907, the compounds of the present invention displaysurprisingly useful properties as antagonists of the CX₃CR1 receptor.

DISCLOSURE OF THE INVENTION

The present invention provides compounds of formula (I)

wherein:

R¹ represents CH₃ or CF₃;

R² represents halo, CN or C₁₋₆alkyl;

R³ represents H or CH₃;

R⁴ represents H or CH₃;

s n represents 0, 1 or 2;

as a free base or a pharmaceutically acceptable salt, solvate or solvateof a salt thereof.

In one embodiment of the invention, there is provided compounds offormula (I), wherein n represents 1.

In another embodiment of the invention, there is provided compounds offormula (I), wherein R¹ represents CH₃.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein R² represents halo or CN.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein R² represents F or Cl.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein R² represents CN.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein n represents 1; R¹ represents CH₃; and R²represents F, Cl or CN.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein the pyridine is attached in its 5-position andhas Cl in 2-position.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein the pyridine is attached in its 2-position andhas CN in 4-position.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein the pyridine is attached in its 2-position andhas F in 5-position.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein the pyridine is attached in its 2-position andhas Cl in 5-position.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein the pyridine is attached in its 2-position andhas F in 3-position.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein the pyridine is attached in its 4-position andhas F in 3-position.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein R³ represents H.

In yet another embodiment of the invention, there is provided compoundsof formula (I), wherein R⁴ represents CH₃.

In yet another embodiment of the invention, there is provided compoundsof formula (I), selected from:

5-{[(1S)-1-(5-chloropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;

5-{[(1S)-1-(5-fluoropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;

5-{[1-(3-fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;

5-{[(1S)-1-(3-Fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;

5-{[(1R)-1-(3-Fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;

5-{[(1S)-1-(3-fluoropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;

2-{(1S)-1-[(7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-2-oxo-2,3-dihydro[1,3]thiazolo[4,5-d]pyrimidin-5-yl)thio]ethyl}isonicotinonitrile;

5-{[(1S)-1-(6-chloropyridin-3-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)butyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;and

5-{[(1S)-1-(6-chloropyridin-3-yl)ethyl]thio}-7-[[(1R)-1-(hydroxymethyl)butyl](methyl)amino][1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;

as a free base or a pharmaceutically acceptable salt, solvate or solvateof a salt thereof.

The compounds of formula (I) may exist in stereoisomeric and/ortautomeric forms. It is to be understood that all enantiomers,diastereomers, racemates, tautomers and mixtures thereof are includedwithin the scope of the invention.

When compared to the compounds disclosed in WO 01/25242, the compoundsof the present invention are characterised by the presence of thebranched thioalkylpyridyl group at the 5-position of thethiazolopyrimidine ring system. That is, the compounds of the presentinvention incorporate a R¹ group that is not hydrogen.

In yet another embodiment of the invention, there is provided a compoundaccording to formula 1, or a pharmaceutically acceptable salt thereof,for use as a medicament.

In yet another embodiment of the invention, there is provided a methodof treating, or reducing the risk of neurodegenerative disorders,demyelinating disease, cardio- and cerebrovascular atheroscleroticdisorders, peripheral artery disease, rheumatoid arthritis, pulmonarydiseases such as COPD, asthma or pain, which comprises administering toa person suffering from or susceptible to such a disease or condition, atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

In yet another embodiment of the invention, there is provided a methodof treating, or reducing the risk of multiple sclerosis, which comprisesadministering to a person suffering from or susceptible to such adisease or condition, a therapeutically effective amount of a compoundof formula (I), or a pharmaceutically acceptable salt thereof.

According to the invention, we further provide a process for thepreparation of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, which comprises:

a) reacting a compound of formula (II):

wherein R³ and R⁴ are as defined in formula (I);

with a compound of formula (III):

wherein R¹, R² and n are as defined in formula (I) and L¹ represents aleaving group; or

b) hydrolysing a compound of formula (IV)

wherein R¹, R², R³, R⁴ and n are as defined in formula (I);

and where necessary converting the resultant compound of formula (I), oranother salt thereof, into a pharmaceutically acceptable salt thereof;or converting the resultant compound of formula (I) into a furthercompound of formula (I); and where desired converting the resultantcompound of formula (I) into an optical isomer thereof.

In process (a), the reactants (II) and (III) are coupled together in asuitable organic solvent such as dimethylsulfoxide (DMSO), acetonitrileor 1-methyl-2-pyrrolidinone (NMP). The reaction is optionally performedin the presence of an added organic or inorganic base such astriethylamine, N,N-diisopropylethylamine (DIPEA) or sodium hydride. Thereaction is performed in the presence of a mild reducing agent such asodium borohydride. The reaction is conducted at a suitable temperature,normally between room temperature and the boiling point of the solvent.The reaction is generally continued for a period of about one hour toone week, or until analysis indicates that formation of the requiredproduct is complete. A suitable leaving groups L¹ is halogen,particularly chloro or bromo. In one embodiment, L¹ represents chloro.

In process (b), the reactant (IV) is subjected to acid catalysedhydrolysis in a suitable organic solvent such as 1,4-dioxane,tetrahydrofuran (THF), dimethylsulphoxide (DMSO) or1-methyl-2-pyrrolidinone (NMP). Suitable acids include inorganic acidssuch as hydrochloric acid or hydrobromic acid, or strong organic acidssuch as trifluoroacetic acid. The reaction is conducted at a suitabletemperature, normally between room temperature and the boiling point ofthe solvent. The reaction is generally continued for a period of aboutone hour to one day, or until analysis indicates that formation of therequired product is complete.

It will be apparent to a person skilled in the art that in the aboveprocesses it may be desirable or necessary to protect an amine, hydroxylor other potentially reactive group. Suitable protecting groups anddetails of processes for adding and removing such groups are, ingeneral, well known in the art. See, for example, “Protective Groups inOrganic Synthesis”, 3rd Edition (1999) by Greene and Wuts.

The present invention includes compounds of formula (I) in the form ofsalts. Suitable salts include those formed with organic or inorganicacids or organic or inorganic bases. Such salts will normally bepharmaceutically acceptable although salts of non-pharmaceuticallyacceptable acids or bases may be of utility in the preparation andpurification of the compound in question.

Salts of compounds of formula (I) may be formed by reacting the freecompound, or a salt, enantiomer or racemate thereof, with one or moreequivalents of the appropriate acid or base. The reaction may be carriedout in a solvent or medium in which the salt is insoluble or in asolvent in which the salt is soluble, for example, water, dioxan,ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents,which may be removed in vacuo or by freeze drying. The reaction may alsobe a metathetical process or it may be carried out on an ion exchangeresin.

Compounds of formula (II) may, in general, be prepared using knownmethods that will be readily apparent to the man skilled in the art. Onesuch suitable route is shown in Scheme 1.

Compounds of formulae (III) are either commercially available, or knownin the literature, or may be prepared using known methods that will bereadily apparent to the man skilled in the art.

Compounds of formula (IV) are either known from for example WO 01/25242or WO 05/33115 or may be prepared using known methods that will bereadily apparent to the man skilled in the art. One such suitable routeis shown in Scheme 2.

Compounds of formula (V) are either known from WO 01/58907, WO 01/25242,or WO 02/76990 or may be prepared using known methods that will bereadily apparent to the man skilled in the art.

For example, compounds of formula (V), and thence those of formula (VI),may be prepared as shown in Scheme 3:

Suitable specific methods for the preparation of compounds of formulae(II), (III), (IV), (V) and (VI) are detailed in the Examples section ofthe present application and such methods represent specific embodimentsof the processes of the invention.

Intermediate compounds may be used as such or in protected form.Suitable protecting groups and details of processes for adding andremoving such groups are, in general, well known in the art. See, forexample, “Protective Groups in Organic Synthesis”, 3rd Edition (1999) byGreene and Wuts.

The compounds of the invention and intermediates thereto may be isolatedfrom their reaction mixtures and, if necessary further purified, byusing standard techniques.

The compounds of formula (I) may exist in stereoisomeric forms.Therefore, all enantiomers, diastereomers, racemates and mixturesthereof are included within the scope of the invention. The variousoptical isomers may be isolated by separation of a stereoisomericmixture of the compounds using conventional techniques, for example,fractional crystallisation, or HPLC.

Alternatively, the various optical isomers may be prepared directlyusing optically active starting materials.

The compounds of formula (I) contain two stereogenic centres and maythus exist in four discrete stereoisomeric forms as shown in formulae(Ia) to (Id)

All such four stereoisomers and any mixtures thereof are included withinthe scope of the invention. In one embodiment, the compounds of formula(I) have the stereochemistry shown in formula (Ia). In anotherembodiment, the compounds of formula (I) have the stereochemistry shownin formula (Ib).

Intermediate compounds may also exist in stereoisomeric forms and may beused as purified enantiomers, diastereomers, racemates or mixtures.

In this specification the term “C₁₋₆alkyl” includes both straight andbranched chain as well as cyclic alkyl groups. C₁₋₆alkyl having 1 to 6carbon atoms and may be, but is not limited to, methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl,t-pentyl, neo-pentyl, n-hexyl, i-hexyl or cyclohexyl.

In this specification the term “halo” or “halogen” refers to fluoro,chloro, bromo, and iodo.

The compounds of formula (I), and their pharmaceutically acceptablesalts are useful because they possess pharmacological activity asantagonists of the CX₃CR1 receptor. In particular, when compared to thecompounds specifically exemplified in WO 01/25242, the compounds offormula (I) of the present invention possess significantly improvedpotencies for inhibition of the CX₃CR1 receptor and/or decreasedpotencies for inhibition of the CXCR2 receptor. Preferred compounds ofthe present invention display both enhanced potency for the inhibitionof CX₃CR1 and decreased potency for inhibition of CXCR2.

In one aspect the present invention provides a compound of formula (I)or a pharmaceutically acceptable salt thereof, for use as a medicament.

In another aspect the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatment or prophylaxis ofdiseases or conditions in which antagonism of the CX₃CR1 receptor isbeneficial.

In another aspect the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatment or prophylaxis ofneurodegenerative disorders, demyelinating disease, cardio- andcerebrovascular atherosclerotic disorders, peripheral artery disease,rheumatoid arthritis, pulmonary diseases such as COPD, asthma or pain.

In another aspect the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatment or prophylaxis ofmultiple sclerosis (MS).

In another aspect the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatment or prophylaxis ofatherosclerosis by preventing and/or reducing the formation of newatherosclerotic lesions or plaques and/or by preventing or slowingprogression of existing lesions and plaques.

In another aspect the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatment or prophylaxis ofatherosclerosis by changing the composition of the plaques to reduce therisk of plaque rupture and atherothrombotic events.

In another aspect the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatment or prophylaxis of strokeor transient brain injury (TBI).

According to the invention, there is also provided a method of treating,or reducing the risk of, diseases or conditions in which antagonism ofthe CX₃CR1 receptor is beneficial which comprises administering to aperson suffering from or at risk of, said disease or condition, atherapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof.

There is also provided a method of treating, or reducing the risk of,neurodegenerative disorders, demyelinating disease, cardio- andcerebrovascular atherosclerotic disorders, peripheral artery disease,rheumatoid arthritis, pulmonary diseases such as COPD, asthma or pain ina person suffering from or at risk of, said disease or condition,wherein the method comprises administering to the person atherapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof.

There is also provided a method of treating, or reducing the risk ofmultiple sclerosis (MS) in a person suffering from or at risk of, saiddisease or condition, wherein the method comprises administering to theperson a therapeutically effective amount of a compound of formula (I)or a pharmaceutically acceptable salt thereof.

There is also provided a method of treating, or reducing the risk ofatherosclerosis by preventing and/or reducing the formation of newatherosclerotic lesions or plaques and/or by preventing or slowingprogression of existing lesions and plaques in a person suffering fromor at risk of, said disease or condition, wherein the method comprisesadministering to the person a therapeutically effective amount of acompound of formula (I) or a pharmaceutically acceptable salt thereof.

There is also provided a method of treating, or reducing the risk ofatherosclerosis by changing the composition of the plaques so as toreduce the risk of plaque rupture and atherothrombotic events in aperson suffering from or at risk of, said disease or condition, whereinthe method comprises administering to the person a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof.

In another aspect the invention provides a pharmaceutical formulationcomprising a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier, for use in thetreatment or prophylaxis of diseases or conditions in which antagonismof the CX₃CR1 receptor is beneficial.

In another aspect the invention provides a pharmaceutical formulationcomprising a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier, for use in thetreatment or prophylaxis of neurodegenerative disorders, demyelinatingdisease, cardio- and cerebrovascular atherosclerotic disorders,peripheral artery disease, rheumatoid arthritis, COPD, asthma or pain.

In another aspect the invention provides a pharmaceutical formulationcomprising a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier, for use in thetreatment or prophylaxis of multiple sclerosis.

In another aspect the present invention provides a pharmaceuticalformulation comprising a therapeutically effective amount of a compoundof formula (I), or a pharmaceutically acceptable salt thereof, inadmixture with a pharmaceutically acceptable adjuvant, diluent orcarrier, for use in the treatment or prophylaxis of atherosclerosis bypreventing and to reducing the formation of new atherosclerotic lesionsand/or plaques and/or by preventing or slowing progression of existinglesions and plaques.

The compounds can be used as monotheraphy, or in combinations, either asprophylactic or therapheutic treatment of inflammatory conditions anddiseases of the central nervous system such as stroke and transientbrain injury (TBI). (Soriano et al. J. Neuroimmunology 2002, 125,59-65.).

In another aspect the present invention provides a pharmaceuticalformulation comprising a therapeutically effective amount of a compoundof formula (I), or a pharmaceutically acceptable salt thereof, inadmixture with a pharmaceutically acceptable adjuvant, diluent orcarrier, for use in the treatment or prophylaxis of atherosclerosis bychanging the composition of the plaques so as to reduce the risk ofplaque rupture and atherothrombotic events.

The compounds of formula (I) and their pharmaceutically acceptable saltsare indicated for use in the treatment or prophylaxis of diseases orconditions in which modulation of activity at the CX₃CR1 receptor isdesirable. In particular, the compounds are indicated for use in thetreatment of neurodegenerative disorders or demyelinating disease inmammals including man. More particularly, the compounds are indicatedfor use in the treatment of multiple sclerosis. The compounds are alsoindicated to be useful in the treatment of pain, rheumatoid arthritis,osteoarthritis, cardio- and cerebrovascular atherosclerotic disorders,peripheral artery disease and pulmonary arterial hypertension.

Conditions that may be specifically mentioned are: neurodegenerativediseases and dementia disorders, for example, Alzheimer's disease,amyotrophic lateral sclerosis and other motor neuron diseases,Creutzfeldt-Jacob's disease and other prion diseases, HIVencephalopathy, Huntington's disease, frontotemporal dementia, Lewy bodydementia and vascular dementia; polyneuropathies, for example,Guillain-Barre syndrome, chronic inflammatory demyelinatingpolyradiculoneuropathy, multifocal motor neuropathy and plexopathies;CNS demyelination, for example, acute disseminated/haemorrhagicencephalomyelitis and subacute sclerosing panencephalitis; neuromusculardisorders, for example, myasthenia gravis and Lambert-Eaton syndrome;spinal disorders, for example, tropical spastic paraparesis andstiff-man syndrome; paraneoplastic syndromes, for example, cerebellardegeneration and encephalomyelitis; traumatic brain injury; migraine;cancer; allograft rejection; systemic sclerosis; viral infections;parasite-transmitted diseases, for example, malaria; periodontaldisease; myocardial infarction; stroke; coronary heart disease;ischaemic heart disease; and restenosis; rheumatoid arthritis; pulmonarydiseases such as COPD; asthma or pain.

The compounds of the invention are also indicated for use in thetreatment of atherosclerosis by preventing and/or reducing the formationof new atherosclerotic lesions or plaques and/or by preventing orslowing progression of existing lesions and plaques.

The compounds of the invention are also indicated for use in thetreatment of atherosclerosis by changing the composition of the plaquesso as to reduce the risk of plaque rupture and atherothrombotic events.

The compounds of the invention are also indicated for use in thetreatment of inflammatory bowel disease (IBD), for example, Crohn'sdisease and ulcerative colitis, by inducing remission and/or maintainingremission of IBD.

Prophylaxis is expected to be particularly relevant to the treatment ofpersons who have suffered a previous episode of, or are otherwiseconsidered to be at increased risk of, the disease or condition inquestion. Persons at risk of developing a particular disease orcondition generally include those having a family history of the diseaseor condition, or those who have been identified by genetic testing orscreening to be particularly susceptible to developing the disease orcondition.

For the above mentioned therapeutic indications, the dosage administeredwill, of course, vary with the compound employed, the mode ofadministration and the treatment desired. However, in general,satisfactory results are obtained when the compounds are administered ata dosage of the solid form of between 1 mg and 2000 mg per day.

The compounds of formula (I) and pharmaceutically acceptable derivativesthereof, may be used on their own, or in the form of appropriatepharmaceutical compositions in which the compound or derivative is inadmixture with a pharmaceutically acceptable adjuvant, diluent orcarrier. Administration may be by, but is not limited to, enteral(including oral, sublingual or rectal), intranasal, intravenous, topicalor other parenteral routes. Conventional procedures for the selectionand preparation of suitable pharmaceutical formulations are describedin, for example, “Pharmaceuticals—The Science of Dosage Form Designs”,M. E. Aulton, Churchill Livingstone, 1988. The pharmaceuticalcomposition preferably comprises less than 80% and more preferably lessthan 50% of a compound of formula (I), or a pharmaceutically acceptablesalt thereof.

There is also provided a process for the preparation of such apharmaceutical composition that comprises mixing the ingredients.

There is also provided a process for the preparation of such apharmaceutical composition that comprises mixing the ingredients.

The invention further relates to combination therapies wherein acompound of formula (I) or a pharmaceutically acceptable salt thereof,or a pharmaceutical composition or formulation comprising a compound offormula (I), is administered concurrently or sequentially with therapyand/or an agent for the treatment of any one of cardio- andcerebrovascular atherosclerotic disorders and peripheral artery disease.

In particular, a compound of formula (I) or a pharmaceuticallyacceptable salt thereof may be administered in association withcompounds from one or more of the following groups:

1) anti-inflammatory agents, for example,

-   -   a) NSAIDs (e.g. acetylsalicylic acid, ibuprofen, naproxen,        flurbiprofen, diclofenac, indometacin);    -   b) leukotriene synthesis inhibitors (5-LO inhibitors e.g.        AZD4407, Zileuton, licofelone, CJ13610, CJ13454; FLAP inhibitors        e.g. BAY-Y-1015, DG-031, MK591, MK886, A81834; LTA4 hydrolase        inhibitors e.g. SC56938, SC57461A);    -   c) leukotriene receptor antagonists; (e.g. CP195543, amelubant,        LY293111, accolate, MK571);

2) anti-hypertensive agents, for example,

-   -   a) beta-blockers (e.g. metoprolol, atenolol, sotalol);    -   b) angiotensin converting enzyme inhibitors (e.g. captopril,        ramipril, quinapril, enalapril);    -   c) calcium channel blockers (e.g. verapamil, diltiazem,        felodipine, amlodipine);

1d) angiotensin II receptor antagonists (e.g. irbesartan, candesartan,telemisartan, losartan);

3) anti-coagulantia, for example,

-   -   a) thrombin inhibitors (e.g. ximelagatran), heparines, factor Xa        inhibitors;    -   b) platelet aggregation inhibitors (e.g. clopidrogrel,        ticlopidine, prasugel, AZ4160);

4) modulators of lipid metabolism, for example,

-   -   a) insulin sensitizers such as PPAR agonists (e.g. pioglitazone,        rosiglitazone, Galida, muraglitazaar, gefemrozil, fenofibrate);    -   b) HMG-CoA reductase inhibitors, statins (e.g. simvastatin,        pravastatin, atorvaststin, rosuvastatin, fluvastatin,        pitavastatin);    -   c) cholesterol absorption inhibitors (e.g. ezetimibe);    -   d) IBAT inhibitors (e.g. AZD-7806);    -   e) LXR agonists (e.g. GW-683965A, T-0901317);    -   f) FXR receptor modulators;    -   g) phospholipase inhibitors;

5) anti-anginal agents, for example, nitrates and nitrites;

6) modulators of oxidative stress, for example, anti-oxidants.(probucol), myeloperoxidase inhibitors.

The invention is illustrated, but in no way limited, by the followingexamples:

General Methods

All solvents used were analytical grade and commercially availableanhydrous solvents were routinely used for reactions. Reactions weretypically run under an inert atmosphere of nitrogen or argon.

¹H and ¹³C NMR spectra were recorded at 400 MHz for proton and 100 MHzfor carbon-13 either on a Varian Unity+ 400 NMR Spectrometer equippedwith a 5 mm BBO probe with Z-gradients, or a Bruker Avance 400 NMRspectrometer equipped with a 60 μl dual inverse flow probe withZ-gradients, or a Bruker DPX400 NMR spectrometer equipped with a4-nucleus probe equipped with Z-gradients. 600 MHz ¹H NMR spectra wererecorded on a Bruker av600 NMR spectrometer equipped with a 5 mm BBIprobehead with Z-gradients. 300 MHz ¹H NMR spectra were recorded on aVarian Gemini 300 NMR equipped with a 5 mm BBI probehead. 500 MHz ¹H NMRspectra were recorded on a Varian Inova 500 Spectrometer operating at amagnetic field of 11.74 T, equipped with a 5 mm nuclei gradient probe.Unless specifically noted in the examples, spectra were recorded at 400MHz for proton and 100 MHz for carbon-13. The following referencesignals were used: the middle line of DMSO-d₆ δ 2.50 (¹H), δ 39.51(¹³C); the middle line of CD₃OD δ 3.31 (¹H) or δ 49.15 (¹³C); acetone-d₆2.04 (¹H), 206.5 (¹³C); and CDCl₃ δ 7.26 (¹H), the middle line of CDCl₃δ 77.16 (¹³C) (unless otherwise indicated).

Enantiomeric excess (ee) was determined by GC on a Cyclodex B column(isothermic elution 100° C.) or on a Cyclosil B column (temperaturegradient 110-130° C.). Diastereomeric excess (de) was determined byHPLC.

Mass spectra were recorded on a Waters LCMS consisting of an Alliance2795 (LC) and a ZQ single quadrupole mass spectrometer. The massspectrometer was equipped with an electrospray ion source (ESI) operatedin a positive or negative ion mode. The capillary voltage was 3 kV andthe mass spectrometer was scanned from m/z 100-700 with a scan time of0.3 or 0.8 s. Separations were performed on either Waters X-Terra MS,C8-columns, (3.5 μm, 50 or 100 mm×2.1 mm i.d.), or a ScantecLab's ACE 3AQ column (100 mm×2.1 mm i.d.). The column temperature was set to 40° C.A linear gradient was applied using a neutral or acidic mobile phasesystem, running at 0% to 100% organic phase in 4-5 minutes, flow rate0.3 ml/min. Neutral mobile phase system: acetonitrile/[10 mM NH₄OAc(aq.)/MeCN (95:5)], or [10 mM NH₄OAc (aq.)/MeCN (1/9)]/[10 mM NH₄OAc(aq.)/MeCN (9/1)]. Acidic mobile phase system: [133 mM HCOOH (aq.)/MeCN(5/95)]/[8 mM HCOOH (aq.)/MeCN (98/2)]. Alternatively, mass spectra wererecorded on a Micromass LCT mass spectrometer equipped with anelectrospray ion source (ESI) operated in a positive ion mode.

Compound identification was performed on a GC-MS (GC 6890, 5973N MSD)supplied by Agilent Technologies. The column used was a VF-5 MS, ID 0.25mm×30 m, 0.25 μm (Varian Inc.). A linear temperature gradient wasapplied starting at 40° C. (hold 1 min) and ending at 300° C. (hold 1mM), 25° C./minute. The MS was equipped with an EI ion source. The MSwas scanned between m/z 50-500 and the scan speed was set to 3.25scan/s. The electron voltage was set to 70 eV.

HPLC analyses were performed on an Agilent HP1000 system consisting ofG1379A Micro Vacuum Degasser, G1312A Binary Pump, G1367A Wellplateauto-sampler, G1316A Thermostatted Column Compartment and G1315B DiodeArray Detector.

Column: X-Terra MS, Waters, 4.6×50 mm, 3.5 μm. The column temperaturewas set to 40° C. and the flow rate to 1.5 ml/min. The Diode ArrayDetector was scanned from 210-300 nm, step and peak width were set to 2nm and 0.05 min, respectively. A linear gradient was applied, run from0% to 100% acetonitrile, in 4 mM Mobile phase: acetonitrile/10 mMammonium acetate in 5% acetonitrile in MilliQ Water.

A typical workup procedure after a reaction consisted of extraction ofthe product with a solvent such as ethyl acetate, washing with waterfollowed by drying of the organic phase over MgSO₄ or Na₂SO₄, andconcentration of the solution in vacuo. Thin layer chromatography (TLC)was performed on Merck TLC-plates (Silica gel 60 F₂₅₄) and UV was usedto visualize the spots. Flash chromatography was preformed on a CombiFlash® Companion™ using RediSep™ normal-phase flash columns or on MerckSilica gel 60 (0.040-0.063 mm). Typical solvents used for flashchromatography were mixtures of chloroform/methanol, toluene/ethylacetate and ethyl acetate/hexanes.

Preparative chromatography was run on a Gilson auto-preparative HPLCwith a diode army detector using a XTerra MS column (C8, 19×300 mm, 7μm), and a gradient with acetonitrile/0.1M ammonium acetate in 5%acetonitrile in MilliQ Water, run from 20% to 60% acetonitrile, in 13mM, and a flow rate of 20 ml/min , unless stated otherwise in theexamples. Alternatively, purification was achieved on a semi preparativeShimadzu LC-8A HPLC with a Shimadzu SPD-10A UV-vis.-detector equippedwith a Waters Symmetry® column (C 18, 5 μm, 100 mm×19 mm). Gradient withacetonitrile/0.1% trifluoroacetic acid in MilliQ Water, run from 35% to60% acetonitrile in 20 min. Flow rate: 10 ml/min Alternativelypreparative HPLC was run on a Agilent 1100 Instrument with UV detection.Column: Kromasil-C18, 20×250 mm, 10 μm. Isocratic elution with mobilephase acetonitrile/MilliQ Water/Formic acid (46/54/0.1). Flow rate: 19ml/min.

Recrystallization was typically performed in solvents or solventmixtures such as ether, ethyl acetate/heptanes and methanol/water.

The following abbreviations have been used: DCM=dichloromethane;de=diastereomeric excess; DIPC1=β-chlorodiisopinocamphenylborane(DIP-Chloride™); DIPEA=N,N-diisopropylethylamine;DMF=N,N-dimethylformamide; DMSO=dimethylsulfoxide; ee=enantiomericexcess; NCS=N-chlorosuccinimide; NMP=1-methyl-2-pyrrolidinone;THF=tetrahydrofuran; aq=aqueous; conc=concentrated.

Starting materials used were either available from commercial sources orprepared according to literature procedures and had experimental data inaccordance to those reported. The following are examples of startingmaterial that were prepared:

(2R)-2-[(2-amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol:WO 02/076990 (Examples 1-4);

5-(benzylthio)-7-chloro[1,3]thiazolo[4,5-d]pyrimidin-2-amine: WO00/09511 (Examples 6 and 7);

5-Fluoro-pyridine-2-carbonitrile: WO 2005/066155 (Example 2);

1-(3-fluoropyridin-4-yl)ethanol: Marsais, F. et al. Tetrahedron 1983,39, 2009-2021 (Example 3);

2-Acetyl-isonicotinonitrile: Citterio et al. J. Chem. Res. Synopses1982, 10, 272-273 (Example 5);

1-(6-Chloropyridin-3-yl)ethanone: Lee, C. et al. J. Med. Chem. 2001, 44,2133 (Examples 6 and 7).

In the general methods that follow, R³ and R⁴ are as defined in formula(I); Py represents an optionally substituted pyridyl, and LG representsa leaving group.

General Method A

Sodium borohydride (0.1 equiv.), DIPEA (1.5 equiv.) and (III) (1.2equiv.) were added (V) (1.0 equiv.) in DMSO under a nitrogen atmosphere.The resulting reaction mixture was stirred at 40° C. until the reactionwas complete (monitored by LC-MS, HPLC or TLC). The mixture was pouredinto ice water and the product was extracted with DCM or EtOAc. Thecombined organic phases were dried and concentrated in vacuo. The crudeproduct was, if necessary, purified using preparative HPLC or by flashcolumn chromatography.

General Method B

Conc. HCl (2.5 mL/mmol (VI)) was added to (VI) (1.0 equiv.) in CH₃CN.The reaction mixture was cooled in an ice bath and sodium nitrite (2.0equiv.) dissolved in a minimal amount of water was added dropwise. Thereaction was stirred at 0° C. until the reaction was complete (monitoredby LC-MS, HPLC or TLC) and was then poured into ice water, neutralizedwith sodium bicarbonate and extracted with DCM or EtOAc. The combinedorganic phases were dried and concentrated in vacuo to give the product.

General Method C

Potassium hydroxide (2.0 equiv.) dissolved in methanol was addeddropwise to a cooled (0° C.) solution of (VII) (1.0 equiv.) in methanol.The resulting mixture was stirred at 0° C. until the reaction wascomplete (monitored by LC-MS, HPLC or TLC). The solvent was evaporatedoff and the product was used in the next reaction step without furtherpurification.

General Method D

A solution of concentrated HCl (1.0 equiv.) was added to a cooled (0°C.) solution of (IV) (1.0 equiv.) in 1,4-dioxane. The resulting mixturewas stirred at 40° C. until the reaction was complete (monitored byLC-MS, HPLC or TLC). The reaction mixture was neutralised with saturatedNaHCO₃ (aq) and the dioxane was evaporated off. The residue wasdissolved in DCM or EtOAc, washed with brine, dried and concentrated invacuo. The crude product was, if necessary, purified using preparativeHPLC or by flash column chromatography.

General Method E1

(VIII) (1.0 equiv.) in THF was added at 0° C. to (+)-DIPC1 (to give(IX)) or (−)-DIPC1 (to give (X)) (1.5 equiv.) in THF under an argonatmosphere. The reaction mixture was allowed to slowly reach roomtemperature overnight. The solvent was evaporated off followed by theaddition of Et₂O and diethanolamine (2.2 equiv.). The mixture wasstirred until the reaction was complete (monitored by LC-MS, HPLC orTLC). The precipitate that formed was filtered off, washed with Et₂O andthe filtrate was concentrated in vacuo. The crude product was, ifnecessary, purified using preparative HPLC or by flash columnchromatography.

General Method E2

(R)-(+)-2-methyl-CBS-oxazaborolidine (1M in toluene, 0.1-1 equiv.) wasdissolved in THF and cooled to 0° C. Borane-methyl sulfide complex (2Min THF, 1 equiv.) was added dropwise and the reaction mixture wasstirred for 1 h. The reaction mixture was cooled to −10° C. and (VIII)(1 equiv.), dissolved in THF was added dropwise over 0.5 h. Theresulting mixture was stirred for 1 h, or until the reaction wascomplete, and the temperature was slowly raised to 10° C. 1M HCl aq. wasadded to quench the reaction. Saturated NaHCO₃ aq. was added until pHwas approximately 8. The product was extracted with DCM. The combinedorganic extracts were dried over Na₂SO₄ and concentrated in vacuo toyield (X). The product was optionally purified by column chromatography.

General Method F1

Triphenyl phosphine (1.3 equiv.) in THF was added at 0° C. to NCS (1.3equiv.) in THF under an argon atmosphere. The resulting mixture wasstirred at ambient temperature for 30 min. (IX) or (X) (1 equiv.) wasadded at 0° C. and the reaction mixture was stirred at ambienttemperature until the reaction was complete (monitored by LC-MS, HPLC orTLC). The solvent was evaporated off followed by addition of hexane andremoval of the precipitate by filtration. The filtrate was concentratedin vacuo and the crude product was, if necessary, purified usingpreparative HPLC or by flash column chromatography.

General method F2

Cyanuric chloride (0.6 equiv.) was dissolved in ethyl acetate. DMF (1.5equiv.) was added and the mixture was stirred at room temperature for 10min. The reaction mixture was cooled to 0° C. (IX) or (X) (1 equiv.) wasdissolved in ethyl acetate and added dropwise during 10 min. Theresulting mixture was stirred at room temperature over night.Isopropanol (ca 0.25 mL/mmol (IX) or (X)) was added. The precipitate wasfiltered off and washed with EtOAc. The filtrate was concentrated toyield (XI) or (XII).

General Method G

Sodium borohydride (1 to 2 equiv.) was added to (II) (1.0 equiv.) inDMSO. Once effervescence had ceased, (III) (2-2.5 equiv.) was added. Theresulting reaction mixture was stirred at 40° C. until the reaction wascomplete (monitored by LC-MS, HPLC or TLC). Purification, if necessary,was achieved using preparative HPLC or by flash column chromatography.

EXAMPLE 1 5-{[(1S)-1-(5-Chloropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

a) 1-(5-Chloropyridin-2-yl)ethanone

5-Chloropyridine-2-carbonitrile (10.71 g, 77 mmol) was dissolved indiethylether (65 mL) and THF (35 mL) under a nitrogen atmosphere. Themixture was cooled until the internal temperature was −63° C. Methylmagnesium bromide (3M in THF, 35 mL, 105 mmol) was added over 30 min.The reaction mixture was then left stirring at −60° C. for 45 min andwas then warmed to room temperature. 50 mL of THF was added to dissolveany precipitated material. After 1 h at room temperature the reactionwas judged complete by HPLC. 2M hydrochloric acid (aq., 100 mL) wasadded and the reaction mixture was stirred for 4 h. pH was adjusted to 7with sodium bicarbonate. The phases were separated and the productextracted from the aqueous phase twice with DCM. The combined organiscextracts were dried over sodium sulphate and concentrated in vacuo. Theproduct was purified by column chromatography (eluent heptane: ethylacetate gradient) to yield 7.9 g (64% yield) of the title compound.

¹H NMR (300 MHz, CDCl₃) δ ppm 8.62 (m, 1H); 8.00 (m, 1H); 7.80 (m, 1H);2.70 (s, 3H).

b) (1S)-1-(5-Chloropyridin-2-yl)ethanol

The title compound was prepared by General method E2 starting from1-(5-chloropyridin-2-yl)ethanone (780 mg, 5 mmol). Purification by flashcolumn chromatography yielded 695 mg (88% yield) of the title compoundwith 92% ee.

¹H NMR (300 MHz, CDCl₃): 8.47 (s, 1H); 7.65 (d, 1H); 7.26 (d, 1H); 4.87(q, 1H); 3.87 (br s, 1H); 1.47 (d, 3H); MS (ESI) m/z 140 and 142 [M+1]⁺.

c) 5-Chloro-2-[(1R)-1-chloroethyl]pyridine

The title compound was prepared by General method F2 starting from(1S)-1-(5-chloropyridin-2-yl)ethanol (695 mg, 4.41 mmol). The crudeproduct was used in the next step without purification.

¹H NMR (400 MHz, CDCl₃): δ ppm 8.46 (d, 1H), 7.64 (dd, 1H), 7.41 (d,1H), 5.08 (q, 1H), 1.80 (d, 3H); MS (ESI) m/z 176 and 178 [M+1]⁺.

d)(2R)-2-[(2-Amino-5-{[(1S)-1-(5-chloropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared by general method A starting from(2R)-2-[(2-amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(823 mg, 2.75 mmol) and 5-chloro-2-[(1R)-1-chloroethyl]pyridine (<4.4mmol). Purification by flash column chromatography (eluent DCM: methanolgradient) yielded 350 mg (30% yield) of the title compound.

¹H NMR (400 MHz, CD₃OD): δ ppm 8.49 (d, 1H), 7.79 (dd, 1H), 7.66 (d,1H), 5.22 (q, 1H), 4.46 (br s, 1H), 3.40-3.57 (m, 2H), 1.66-1.78 (m,4H), 1.40-1.61 (m, 2H), 0.93-1.03 (m, 6H); MS (ESI) m/z 439 and 441[M+1]⁺.

e)(2R)-2-[(2-Chloro-5-{[(1S)-1-(5-chloropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared by general method B starting from(2R)-2-[(2-amino-5-{[(1S)-1-(5-chloropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(340 mg, 0.77 mmol). MS (ESI) m/z 458 and 460 [M+1]⁺.

f)(2R)-2-[(5-{[(1S)-1-(5-Chloropyridin-2-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared from(2R)-2-[(2-chloro-5-{[(1S)-1-(5-chloropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olfrom the previous step using General method C, except that the reactionmixture was heated to 50° C. for 1 h. After complete reaction thereaction mixture was diluted with water and the product was extractedwith DCM (four times). The combined organic extracts were dried oversodium sulphate and concentrated in vacuo to yield the title compoundthat was used in the next step without purification. MS (ESI) m/z 453and 455 [M+1]⁺.

g)5-{[(1S)-1-(5-Chloropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

The title compound was prepared from(2R)-2-[(5-{[(1S)-1-(5-chloropyridin-2-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olfrom the previous step using General method D except that the reactionmixture was stirred at 50° C. for 2.5 h and then at room temperatureover night. After complete reaction the reaction mixture was dilutedwith Brine and extracted with DCM (three times). The combined organicextracts were dried over sodium sulphate and concentrated in vacuo.

The product was purified by flash chromatography (eluent DCM: methanolgradient) to yield 160 mg. Further purification by preparative HPLC(Column: Chiralcel OJ, eluent: ethanol/heptane 30/70, flow:12 ml/min)yielded 82 mg of the title compound.

¹H NMR (400 MHz, CD₃OD): δ ppm 8.24 (d, 1H), 7.56 (dd, 1H), 7.38 (d,1H), 4.90 (q, 1H), 4.19 (br s, 1H), 3.16-3.30 (m, 2H), 1.39-1.51 (m,4H), 1.15-1.34 (m, 2H), 0.68-0.76 (m, 6 11); ¹H NMR (DMSO-d₆) δ ppm12.36 (br s, 1H), 8.57 (d, 1H), 7.86 (dd, 1H); 7.57 (d, 1H); 7.23 (d,1H); 5.03 (q, 1H); 4.69 (t, 1H); 4.29 (br s, 1H); 3.40-3.25 (m, 2H),1.66 (d, 3H), 1.63-1.52 (m, 1H); 1.48-1.32 (m, 2H), 0.88 (d, 3H), 0.85(d, 3H); MS (ESI) m/z 440 and 442 [M+1]⁺, 438 and 440 [M−1]⁺.

EXAMPLE 25-{[(1S)-1-(5-Fluoropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

a) 1-(5-Fluoropyridin-2-yl)ethanone

5-Fluoro-pyridine-2-carbonitrile (29 g, 240 mmol) was dissolved in THF(150 mL) under a nitrogen atmosphere. The reaction mixture was cooled toan internal temperature of −64° C. Methyl magnesium bromide (3M in THF,105 mL, 315 mmol) was added over 40 min. The reaction mixture wasstirred at −65° C. for 1.5 h, then it was warmed to room temperature.THF (50 mL) was added and the mixture was stirred an additional 3 h. 2Mhydrochloric acid (aq., 100 mL) was added until the mixture was slightlyacidic and the reaction mixture was stirred at room temperature overnight. Sodium bicarbonate was then added to neutralize the reactionmixture. The phases were separated and the aqueous phase was extractedwith DCM. The combined organic extracts were washed with Brine, driedover sodium sulphate and concentrated in vacuo. The crude product waspurified by flash column chromatography to yield 18 g (55% yield) of thetitle compound.

¹H NMR (300 MHz, CDCl₃): 8.50 (m, 1H); 8.10 (m, 1H); 7.52 (m, 1H); 2.70(s, 3H).

b) (1S)-1-(5-Fluoropyridin-2-yl)ethanol

The title compound was prepared by General method E2 starting from1-(5-fluoropyridin-2-yl)ethanone (3.18 g, 22.9 mmol). Purification byflash column chromatography yielded 2.73 g (84% yield) of the titlecompound with 84% ee.

¹H NMR (300 MHz, CDCl₃): 8.38 (m, 1H); 7.5-7.2 (m, 2H); 4.89 (q, 1H);3.9 (br s, 1H); 1.49 (d, 3H).

c) 2-[(1R)-1-Chloroethyl]-5-fluoropyridine

The title compound with 80% ee was prepared by General method F2starting from (1S)-1-(5-fluoropyridin-2-yl)ethanol (720 mg, 5.1 mmol).The crude product was used in the next step without purification.

¹H NMR (300 MHz, CDCl₃): 8.44-8.40 (m, 1H); 7.6-7.4 (m, 2H); 5.16 (q,1H), 1.86 (d, 3H).

d)(2R)-2-[(2-Amino-5-{[(1S)-1-(5-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared by General method A starting from(2R)-2-[(2-amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(940 mg, 3.1 mmol) and 2-[(1R)-1-chloroethyl]-5-fluoropyridine (0.81 g,5.1 mmol). The product was purified by flash column chromatography toyield 0.75 g (56% yield) of the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.51 (d, 1H), 7.98 (s, 2H), 7.65 (dt,1H); 7.58 (dd, 1H), 6.88 (d, 1H); 5.12 (q, 1H); 4.66 (t, 1H); 4.27 (brs, 1H); 3.41-3.27 (m, 2H), 1.66 (d, 3H), 1.65-1.55 (m, 1H); 1.48-1.35(m, 2H), 0.88 (d, 3H), 0.85 (d, 3H); MS (ESI) m/z 423 [M+1]⁺.

e)(2R)-2-[(2-Chloro-5-{[(1S)-1-(5-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared using General method B starting from(2R)-2-[(2-amino-5-{[(1S)-1-(5-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(750 mg, 1.77 mmol). MS (ESI) m/z 442 and 444 [M+1]⁺.

f)(2R)-2-[(5-{[(1S)-1-(5-Fluoropyridin-2-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared from(2R)-2-[(2-chloro-5-{[(1S)-1-(5-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olfrom the previous step using General method C, except that the reactionmixture was heated to 50° C. for 1.5 h. After complete reaction thereaction mixture was diluted with water and Brine and the product wasextracted with chloroform (three times). The combined organic extractswere dried over magnesium sulphate and concentrated in vacuo to yieldthe title compound that was used without purification. MS (ESI) m/z 438[M+1]⁺.

g)5-{[(1S)-1-(5-Fluoropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

The title compound was prepared from(2R)-2-[(5-{[(1S)-1-(5-fluoropyridin-2-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olfrom the previous step using General method D, except that the reactionmixture was stirred at 50° C. for 3 h. After complete reaction thereaction mixture was diluted with Brine and extracted with DCM (threetimes). The combined organic extracts were dried over magnesium sulphateand concentrated in vacuo. The product was purified by flashchromatography (eluent DCM: methanol gradient). Further purification bypreparative HPLC (column Chiralcel OJ, eluent: ethanol, flow: 8 mL/min)yielded 113 mg of the title compound.

¹H NMR (CD₃OD): δ ppm 8.19 (d, 1H), 7.46 (dd, 1H), 7.36 (dt, 1H), 4.97(q, 1H), 4.26 (br s, 1H), 3.23-3.34 (m, 2H), 1.44-1.55 (m, 4H),1.19-1.37 (m, 2H), 0.75 (dd, 6H); ¹H NMR (DMSO-d₆) δ ppm 12.36 (br s,1H), 8.52 (d, 1H), 7.66 (dt, 1H); 7.60 (dd, 1H), 7.23 (d, 1H); 5.07 (q,1H); 4.69 (t, 1H); 4.30 (br s, 1H); 3.40-3.26 (m, 2H), 1.67 (d, 3H),1.64-1.53 (m, 1H); 1.48-1.33 (m, 2H), 0.88 (d, 3H), 0.85 (d, 3H); MS(ESI) m/z 424 [M+1]⁺. OK

EXAMPLE 35-{[1-(3-Fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

a) 4-(1-Chloroethyl)-3-fluoropyridine

1-(3-fluoropyridin-4-yl)ethanol (0.8 g, 5.7 mmol) was treated withthionyl chloride (5 mL) and the resulting mixture was heated to 80° C.for 2 h. Water (10 mL) and sat. sodium bicarbonate (aq., 10 mL) wasadded. The product was extracted with DCM (three times). The combinedorganic extracts were washed with brine, dried over sodium sulphate andconcentrated in vacuo. The crude product was purified by flash columnchromatography (eluent heptane: ethyl acetate gradient) to yield 0.36 g(39% yield) of the title compound.

¹H NMR (300 MHz, CDCl₃) 8.45 (m, 2H), 7.50 (m, 1H), 5.34 (q, 1H), 1.83(d, 3H).

b)(2R)-2-[(2-Amino-5-{[1-(3-fluoropyridin-4-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound (370 mg, 47% yield) was prepared using General methodA starting from(2R)-2-[(2-amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(560 mg, 1.87 mmol). MS (ESI) m/z 423 [M+1]⁺.

c)(2R)-2-[(2-Chloro-5-{[1-(3-fluoropyridin-4-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared using General method B starting from(2R)-2-[(2-amino-5-{[1-(3-fluoropyridin-4-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(370 mg, 0.84 mmol).

d)(2R)-2-[(5-{[1-(3-Fluoropyridin-4-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared from(2R)-2-[(2-chloro-5-{[1-(3-fluoropyridin-4-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olfrom the previous step using General method C, except that the reactionmixture was heated to 50° C. for 1.5 h. After complete reaction thereaction mixture was diluted with water and Brine (1:1) and the productwas extracted with DCM (twice). The pH of the water phase was thenadjusted to 7 with ammonium chloride and the product was extracted withDCM (twice). The combined organic extracts were dried over sodiumsulphate and concentrated in vacuo to yield the title compound.

e)5-{[1-(3-Fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

The title compound was prepared starting from(2R)-2-[(5-{[1-(3-fluoropyridin-4-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olfrom the previous step using General method D, except that the reactionmixture was stirred at 50° C. for 2 h. After complete reaction thereaction mixture was diluted with sat. sodium bicarbonate aq. and water(1:1) and extracted with DCM (three times). The combined organicextracts were dried over sodium sulphate and concentrated in vacuo. Theproduct was purified by flash column chromatography (eluentheptane:ethyl acetate gradient) to yield the title compound as a mixtureof diastereomers (194 mg). MS (ESI) m/z 424 [M+1]⁺.

EXAMPLE 45-{[(1S)-1-(3-Fluoropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

a) 1-(6-Bromo-3-fluoro-pyridin-2-yl)ethanone

2-Bromo-5-fluoro-pyridine (11 g, 62.5 mmol) was dissolved in diethylether at room temperature under a nitrogen atmosphere. The reactionmixture was cooled until the internal temperature was −66° C. Butyllithium (2.5 M in hexanes, 26 mL, 65 mmol) was added dropwise over 0.5h. The resulting reaction mixture was left at −65° C. for 1 h.N,N-Dimethylacetamide (6.5 mL, 70 mmol) was added over 10 min. and thereaction mixture was stirred at −65° C. for 2 h. 1M hydrochloric acidaq. (50 mL) was added and the mixture was warmed to room temperature.The pH was adjusted to 7 with additional hydrochloric acid. The aqueousphase was extracted with diethyl ether three times. The combined organicphases were washed with Brine, dried over sodium sulphate, andconcentrated in vacuo. Purification by flash column chromatography(eluent heptane:diethyl ether gradient) yielded 4.6 g (34% yield) of thetitle compound.

¹H NMR (300 MHz, DMSO-d₆): 8.0-7.8 (m, 2H); 2.57 (s, 3H); MS (ESI) m/z218 and 220 [M+1]⁺.

b) (1S)-1-(6-Bromo-3-fluoro-pyridin-2-yl)ethanol

The title compound was prepared using General method E2 starting from1-(6-bromo-3-fluoro-pyridin-2-yl)ethanone (1.76 g, 8.19 mmol). Theproduct was purified by flash column chromatography (eluent: heptane:ethyl acetate gradient) to yield 1.31 g (73% yield) of the titlecompound with 80% ee.

¹H NMR (300 MHz, CDCl₃) 7.38 (m, 1H); 7.26 (m, 1H); 5.06 (q, 1H); 3.38(br s, 1H); 1.47 (d, 3H); MS (ESI) m/z 220 and 222 [M+1]⁺, m/z 202[M-H2O]⁺.

c) (1S)-1-(3-Fluoro-pyridin-2-yl)ethanol

(1S)-1-(6-Bromo-3-fluoro-pyridin-2-yl)ethanol (1.3 g, 5.9 mmol),triethylamine (1.6 mL, 11.5 mmol) and palladium on carbon (0.64 g, 0.34mmol) were mixed in DCM (25 mL). The flask was evacuated/filled withhydrogen gas in 4 cycles and then left at 2.5 atm pressure hydrogen gasat room temperature for 24 h. The mixture was filtered and the solidwashed with DCM. The filtrate was washed with water and Brine and driedover sodium sulphate and concentrated in vacuo. The crude product waspurified by flash column chromatography (eluent DCM:methanol gradient)to yield 0.54 g (65% yield) of the title compound.

¹H NMR (300 MHz, CDCl₃): 8.38 (m, 1H); 7.39 (m, 1H); 7.26 (m, 1H); 5.11(q, 1H); 4.16 (br s, 1H); 1.49 (d, 3H).

d) 2-((R)-1-Chloroethyl)-3-fluoro-pyridine

The title compound (0.24 g) was prepared using General method F2starting from (1S)-1-(3-fluoro-pyridin-2-yl)ethanol (254 mg, 1.8 mmol).

¹H NMR (300 MHz, CDCl₃): 8.46 (m, 1H); 7.47 (m, 1H); 7.34 (m, 1H); 5.48(q, 1H), 1.94 (d, 3H); MS (ESI) m/z 160 and 162 [M+1]⁺.

e)(2R)-2-[(2-Amino-5-{[(1S)-1-(3-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared using General method A starting from(2R)-2-[(2-amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(348 mg, 1.16 mmol) and 2-((R)-1-chloroethyl)-3-fluoro-pyridine (240 mg,1.5 mmol). Purification by flash column chromatography (eluent DCM:methanol gradient) resulted in 190 mg (47% yield) of the title compoundwith a diastereomeric excess of 60%.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.40 (dt, 1H), 7.98 (s, 2H), 7.70 (m,1H), 7.40 (m, 1H); 6.92 (d, 1H); 5.45 (q, 1H); 4.65 (t, 1H); 4.27 (br s,1H); 3.45-3.30 (m, 2H), 1.69 (d, 3H), 1.66-1.58 (m, 1H), 1.50-1.35 (m,2H), 0.88 (d, 3H), 0.85 (d, 3H); MS (ESI) m/z 423 [M+1]⁺. MS (ESI) m/z423 [M+1]⁺.

f)(2R)-2-[(2-Chloro-5-{[(1S)-1-(3-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared using General method B starting from(2R)-2-[(2-amino-5-{[(1S)-1-(3-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol(135 mg, 0.32 mmol). MS (ESI) m/z 442 and 444 [M+1]+.

g)(2R)-2-[(5-{[(1S)-1-(3-Fluoropyridin-2-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol

The title compound was prepared from(2R)-2-[(2-chloro-5-{[(1S)-1-(3-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olfrom the next step using General method C, except that the reactionmixture was heated to 50° C. for 1.5 h. After complete reaction thereaction mixture was diluted with water and Brine (2:1) and the productwas extracted with chloroform (three times). The combined organicextracts were dried over magnesium sulphate and concentrated in vacuo toyield the title compound. MS (ESI) m/z 438 [M+1]⁺.

h)5-{[(1S)-1-(3-Fluoropyridin-2-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

The title compound was prepared from(2R)-2-[(5-{[(1S)-1-(3-fluoropyridin-2-yl)ethyl]thio}-2-methoxy[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-olusing General method D, except that the reaction mixture was heated to50° C. for 1.5 h. After complete reaction the reaction mixture wasdiluted with brine and extracted with DCM (three times). The combinedorganic extracts were dried over magnesium sulphate and concentrated invacuo. The product was purified by flash column chromatography (eluentDCM: methanol gradient) followed by preparative HPLC to yield 20 mg ofthe title compound.

¹H NMR (DMSO-d₆) δ ppm 12.37 (br s, 1H), 8.41 (dt, 1H), 7.72 (m, 1H);7.42 (m, 1H); 7.27 (br s, 1H); 5.43 (q, 1H); 4.67 (t, 1H); 4.30 (br s,1H); 3.44-3.30 (m, 2H), 1.70 (d, 3H), 1.65-1.55 (m, 1H); 1.52-1.32 (m,2H), 0.89 (d, 3H), 0.86 (d, 3H); MS (ESI) m/z 424 [M+1]⁺.

EXAMPLE 5 2-{(1S)-1-[(7-{[(1R)-1-(Hydroxymethyl)-3-methylbutyl]amino}-2-oxo-2,3-dihydro[1,3]thiazolo[4,5-d]pyrimidin-5-yl)thiol]ethyl}isonicotinonitrile

a) 2-((S)-1-Hydroxy-ethyl)-isonicotinonitrile

The title compound (1.13 g, 7.63 mmol) was prepared according to GeneralMethod E1 starting from 2-acetyl-isonicotinonitrile (1.42 g, 9.72 mmol)and (−)-DIPC1 (4.67 g, 14.57 mmol).

¹H NMR (500 MHz, CDCl₃) δ 8.72 (d, 1H), 7.62 (s, 1H), 7.44 (dd, 1H),4.96 (q, 1H), 1.54 (d, 3H).

b) 2-((R)-1-Chloro-ethyl)-isonicotinonitrile

The title compound (32.2 mg, 0.19 mmol) was prepared according toGeneral Method F1 starting from2-((S)-1-hydroxy-ethyl)-isonicotinonitrile (400 mg, 2.7 mmol).

¹H NMR (500 MHz, CDCl₃) δ 8.74 (d, 1H), 7.76 (s, 1H), 7.46 (dd, 1H),5.16 (q, 1H), 1.88 (d, 3H).

c)(2R)-2-{2-Chloro-5-[2-chloro-7-((1R)-1-hydroxymethyl-3-methyl-butylamino)-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-thiazolo[4,5-d]pyrimidin-7-ylamino}-4-methyl-pentan-1-ol

Sodium nitrite (5.19 g, 75 mmol) in water (25 mL) was added dropwise at0° C. to(2R)-2-[[2-amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino]-4-methylpentan-1-ol(7.50 g, 25 mmol) in conc. hydrochloric acid (150 mL) and acetonitrile(150 mL). The reaction mixture was stirred for 18 h at 0-5° C., and thenpoured onto ice (500 mL), and extracted with ethyl acetate. Anyremaining solid was filtered off The combined organic phases were washedsequentially with Brine and saturated aqueous sodium bicarbonatesolution. The organic phase was dried and evaporated and the solidpreviously filtered off was added to this. The total solid was slurriedin ethyl acetate, which after filtration provided the title compound(6.3 g, 80% yield).

¹H NMR (DMSO-d₆) δ 8.25 (d, 2H), 4.19 (m, 2H), 3.35 (m, 4H), 1.40 (m,4H), 1.21 (m, 2H), 0.68 (d, 6H), 0.51 (d, 6H); MS (ESI) m/z 635 [M+1]⁺.

d)(2R)-2-{5-[7-((1R)-1-Hydroxymethyl-3-methyl-butylamino)-2-methoxy-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-2-methoxy-thiazolo[4,5-d]pyrimidin-7-ylamino}-4-methyl-pentan-1-ol

Potassium hydroxide (0.53 g, 9.4 mmol) in methanol (5 mL) was added at0° C. to a solution of(2R)-2-{2-chloro-5-[2-chloro-7-((1R)-1-hydroxymethyl-3-methyl-butylamino)-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-thiazolo[4,5-d]pyrimidin-7-ylamino}-4-methyl-pentan-1-ol(3.0 g, 4.7 mmol) in methanol (200 mL). The reaction was maintained at0-5° C. for 18 h. The solvent was evaporated off and the residue takenup in methanol/ethyl acetate (1:1). This solution was rapidlychromatographed (eluent ethyl acetate) to provide is the title compound(2.0 g, 68% yield). MS (ESI) m/z 627 [M+1]⁺.

e)5-[7-{[(1R)-1-(Hydroxymethyl)-3-methylbutyl]amino}-[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one-5-yldisulfanyl]-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

A mixture of conc. hydrochloric acid (20 mL) and water (20 mL) was addedto a solution of(2R)-2-{5-[7-((1R)-1-hydroxymethyl-3-methyl-butylamino)-2-methoxy-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-2-methoxy-thiazolo[4,5-d]pyrimidin-7-ylamino}-4-methyl-pentan-1-ol(1.5 g, 2.4 mmol) in 1,4-dioxane (20 mL). The solution was then stirredat 45° C. for 18 h. The solvent was evaporated off and the residue wastaken up in ethyl acetate. Any undissolved residue was collected byfiltration. The filtrate was subjected to flash column chromatography(eluent ethyl acetate: methanol 95:5). The solid residue and the productcollected from the chromatography were pooled together to give the titlecompound (600 mg, 42% yield).

¹H NMR (DMSO-d₆) δ 12.45 (s, 2H), 7.33 (d, 2H), 4.62 (t, 2H), 4.17 (brs, 2H), 1.48-1.31 (m, 4H), 1.25-1.14 (m, 2H), 0.72 (d, 6H), 0.56 (d,6H); MS (ESI) m/z 599 [M+1]⁺.

f)2-{(1S)-1-[(7-{[(1R)-1-(Hydroxymethyl)-3-methylbutyl]amino}-2-oxo-2,3-dihydro[1,3]thiazolo[4,5-d]pyrimidin-5-yl)thio]ethyl}isonicotinonitrile

The title compound was prepared according to General Method G withaddition of DIPEA (2 equiv.). Starting from5,5′-dithiobis[7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one](64 mg, 0.096 mmol) and 2-((R)-1-chloro-ethyl)-isonicotinonitrile (32mg, 0.192 mmol) the title compound (39 mg) was obtained as adiastereomeric mixture. Purification by preparative HPLC (Column:Kromasil-C18) yielded 15 mg (36% yield) of the title compound with 98%de.

¹H NMR (500 MHz, CD₃OD) δ 8.71 (d, 1H), 7.92 (s, 1H), 7.56 (d, 1H), 5.17(q, 1H), 4.4 (s, 1H), 3.40-3.52 (m, 2H), 1.72 (d 3H), 1.60-1.71 (m 1H),1.38-1.54 (m, 2H), 0.90-0.98 (m 6H); MS (ESI⁺) m/z 431 [M+H]⁺.

EXAMPLE 65-{[(1S)-1-(6-Chloropyridin-3-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)butyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

a)(2R)-2-{[2-Amino-5-(benzylthio)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}pentan-1-ol

5-(Benzylthio)-7-chloro[1,3]thiazolo[4,5-d]pyrimidin-2-amine (6.0 g,19.4 mmol) was dissolved in NMP (30 mL). DIPEA (8.4 mL, 48.5 mmol) and2-amino-(2R)-1-pentanol (3.5 g, 33.9 mmol) were added and the mixturewas heated to 110° C. for 4 days. After cooling to room temperature, themixture was poured into water (200 mL). The precipitated product wascollected by filtration, washed with water and used in the next stepwithout further purification (7.0 g, 97% yield). MS (ESI⁺) m/z 376[M+H]⁺.

b)(2R)-2-[(2-Amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]pentan-1-ol

A round-bottomed flask was equipped with a dry ice-ethanol condenser andimmersed in a dry ice-ethanol cooling bath. Ammonia (250 mL) wascondensed into the flask followed by addition of(2R)-2-{[2-amino-5-(benzylthio)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}pentan-1-ol(6.8 g, 18.1 mmol). The resulting mixture was allowed to warm to −33° C.and sodium metal was added in small pieces until a blue colour appearedand persisted for 30 seconds. The reaction was then quenched by additionof a spoonful of solid ammonium chloride. The ammonia was evaporated offand water (250 mL) was added to the residue. The resulting mixture wasneutralized with 1M hydrochloric acid (aq). The precipitated product wascollected by filtration, washed with water and dried in vacuo to yield4.15 g (80% yield) of the title compound. MS (ESI⁺) m/z 286 [M+H]⁺.

c)(2R)-2-{2-Chloro-5-[2-chloro-7-((1R)-1-hydroxymethylbutylamino)-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-thiazolo[4,5-d]pyrimidin-7-ylamino}-pentan-1-ol

(2R)-2-[(2-Amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]pentan-1-ol(4.0 g, 14 mmol) was dissolved in acetonitrile (100 ml) and concentratedhydrochloric acid (150 mL). Sodium nitrite (1.93 g, 28 mmol) wasdissolved in water (10 mL) and added at 0° C. The reaction mixture wasleft at 0° C. for 2 days until the reaction was complete by LCMS. Thereaction mixture was poured onto ice and the precipitated product wascollected by filtration. The solid was dried in vacuo to give 3.3 g (78%yield) of the title compound.

¹H NMR (DMSO-d₆) δ 8.27 (d, 1H), 4.32-3.81 (m, 2H), 3.50-3.23 (m, 2H),1.37-1.19 (m, 2H), 1.10-0.93 (m, 1H), 0.94-0.78 (m, 1H), 0.49 (t, 3H);MS (ESI) m/z 607 [M+1]⁺.

d)(2R)-2-{5-[7-((1R)-1-Hydroxymethylbutylamino)-2-methoxy-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-2-methoxy-thiazolo[4,5-d]pyrimidin-7-ylamino}-pentan-1-ol

Potassium hydroxide (495 mg, 8.8 mmol) was added to(2R)-2-{2-chloro-5-[2-chloro-7-((1R)-1-hydroxymethylbutylamino)-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-thiazolo[4,5-d]pyrimidin-7-ylamino}-pentan-1-ol(2.68 g, 4.41 mmol) in methanol (200 mL) at 0° C. The reaction wasstirred at 0° C. overnight and then the methanol was evaporated off. Theresidue was poured into water and the resulting precipitate wascollected by filtration. The crude wet product was used in the next stepwithout any further purification. MS (ESI) m/z 599 [M+1]⁺.

e)5-[7-{[(1R)-1-(Hydroxymethyl)]amino}-[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one-5-yldisulfanyl]-7-{[(1R)-1-(hydroxymethylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

Crude(2R)-2-{5-[7-((1R)-1-hydroxymethylbutylamino)-2-methoxy-thiazolo[4,5-d]pyrimidin-5-yldisulfanyl]-2-methoxy-thiazolo[4,5-d]pyrimidin-7-ylamino}-pentan-1-ol(4.41 mmol) from the previous step was dissolved in 1,4-dioxane (100mL). Conc. hydrochloric acid (2 mL) and water (2 mL) were added and theresulting mixture was stirred at 45° C. over night. The solvent wasevaporated in vacuo and the product was precipitated by addition ofwater. The precipitate was collected by filtration and washed withwater. The crude product was purified by flash column chromatography(eluent DCM: ethyl acetate gradient) to give 1.5 g (59% yield over twosteps) of the title compound.

¹H NMR (DMSO-d₆) δ 12.46 (s, 1H), 7.33 (d, 1H), 4.61 (t, 1H), 4.10 (br.s., 1H), 3.35 (t, 2H), 1.37-1.20 (m, 2H), 1.13-1.10 (m, 1H), 0.96-0.82(m, 1H), 0.59 (t, 3H); MS (ESI) m/z 571 [M+1]⁺.

f) (1S)-1-(6-Chloropyridin-3-yl)ethanol

The title compound was prepared in accordance with the General method E1using 1-(6-chloropyridin-3-yl)ethanone (0.80 g, 5.14 mmol), affording0.71 g (88% yield) of the title compound.

¹H NMR (CDCl₃) δ ppm 8.40-8.28 (m, 1H), 7.75-7.63 (m, 1H), 7.35-7.24 (m,1H), 5.04-4.79 (m, 1H), 1.63-1.45 (m, 3H); MS (ESI) m/z 158 and 160[M+1]⁺.

g) 2-Chloro-5-[(1R)-1-chloroethyl]pyridine

The title compound was prepared in accordance with the General method F1using (1S)-1-(6-chloropyridin-3-yl)ethanol (0.20 g, 1.27 mmol),affording 0.16 g (72% yield) of the title compound.

¹H NMR (CDCl₃) δ ppm 8.45-8.35 (m, 1H), 7.79-7.70 (m, 1H), 7.39-7.29 (m,1H), 5.07 (q, 1H), 1.85-1.78 (m, 3H); MS (ESI) m/z 176 and 178 [M+1]⁺.

h)5-{[(1S)-1-(6-Chloropyridin-3-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)butyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

The title compound was prepared in accordance with general method Gusing5-[7-{[(1R)-1-(hydroxymethyl)]amino}-[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one-5-yldisulfanyl]-7-{[(1R)-1-(hydroxymethylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one(0.10 g, 0.175 mmol), 2-chloro-5-[(1R)-1-chloroethyl]pyridine (0.069 g,0.39 mmol) and sodium borohydride (0.040 g, 1.05 mmol), affording 0.055g (37% yield) of the title compound.

¹H NMR (CDCl₃) δ ppm 8.52-8.38 (m, 1H), 7.87-7.72 (m, 1H), 7.30-7.26 (m,1H), 4.91-4.81 (m, 1H), 4.74-4.65 (m, 1H), 4.29-4.17 (m, 1H), 3.68-3.52(m, 2H), 1.69-1.64 (m, 3H), 1.56-1.46 (m, 2H), 1.46-1.32 (m, 2H),0.98-0.90 (m, 3H); MS (ESI) m/z 426 and 428 [M+1]⁺.

EXAMPLE 75-{[(1S)-1-(6-Chloropyridin-3-yl)ethyl]thio}-7-[[(1R)-1-(hydroxymethyl)butyl](methyl)amino][1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

a) N-(Ethoxycarbonyl)-D-norvaline

D-Norvaline (10.0 g, 85.3 mmol) was dissolved in aqueous sodiumhydroxide (4M, 25 mL). Ethyl chloroformate (10.6 mL, 111 mmol) andaqueous sodium hydroxide (4M, 25 mL) was added over 15 min. at 0° C. Thereaction mixture was warmed to room temperature and stirred at thistemperature for 4 h. The reaction mixture was washed with diethyl etherthree times and then acidified with aqueous hydrochloric acid (2M). Theproduct was extracted with diethyl ether three times. The combinedorganic phases were dried over magnesium sulphate and concentrated invacuo to yield the title compound in quantitative yield.

¹H NMR (CDCl₃) δ ppm 6.43 (br s, 1H), 5.22 (d, 1H), 4.37 (q, 1H), 4.13(q, 2H), 1.84 (m, 1H), 1.68 (sextet, 1H), 1.42 (sextet, 1H), 1.25 (t,3H), 0.95 (t, 3H); MS (CI) 144 (100%), 190 [M+1]⁺.

b) (2R)-2-(Methylamino)pentan-1-ol

Lithium aluminium hydride (6.5 g, 171 mmol) was suspended in THF at 0°C. under a nitrogen atmosphere. N-(Ethoxycarbonyl)-D-norvaline wasdissolved in THF and added dropwise at 0° C. The reaction mixture wasrefluxed over night. After cooling to room temperature, saturatedaqueous sodium sulphate was added to form a slurry. The resultingmixture was filtered through celite. The solid was washed with DCM untilall product had been extracted. The combined filtrate was dried oversodium sulphate and concentrated in vacuo. Bulb-to-bulb destillation at0.1 mbar collecting the fraction between 75-85° C. yielded 7.1 g (71%yield) of the title compound.

¹H NMR (CDCl₃) 3.63 (dd, 1H); 3.30 (dd, 1H); 2.51 (m, 1H); 2.41 (s, 3H);2.09 (br s, 2H); 1.50-1.28 (m, 4H); 0.93 (t, 3H); MS (CI) 86 (100%), 118[M+1]⁺.

c)(2R)-2-{[2-Amino-5-(benzylthio)[1,3]thiazolo[4,5-d]pyrimidin-7-yl](methyl)amino}pentan-1-ol

5-(Benzylthio)-7-chloro[1,3]thiazolo[4,5-d]pyrimidin-2-amine (6.0 g,19.4 mmol) was dissolved in NMP (25 mL). DIPEA (6.8 mL, 38.8 mmol) and(2R)-2-(methylamino)pentan-1-ol (3.4 g, 29.1 mmol) were added and themixture was heated to 120° C. for 3 days. Additional(2R)-2-(methylamino)pentan-1-ol (350 mg, 2.99 mmol) and DIPEA (1 mL,5.74 mmol) was added and the reaction mixture was heated for 6 h at 120°C. After cooling to room temperature, the mixture was poured into ice.The precipitated product was collected by filtration and purified byflash column chromatography (eluent DCM: ethyl acetate gradient) toyield the title compound (5.74 g, 76% yield).

¹H NMR (DMSO-d₆) 7.98 (br s, 2H), 7.41 (m, 2H), 7.29 (m, 2H), 7.22 (m,1H), 4.73 (t, 1H), 4.54 (br s, 1H), 4.33 (m, 2H), 3.55-3.40 (m, 2H),3.01 (s, 3H), 1.52-1.44 (m, 2H), 1.25-1.10 (m, 2H), 0.84 (t, 3H); MS(ESI) m/z 390 [M+1]⁺.

d)(2R)-2-[(2-Amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)(methyl)amino]pentan-1-ol

A round-bottomed flask was equipped with a dry ice-ethanol condenser andimmersed in a dry ice-ethanol cooling bath. Ammonia (200 mL) wascondensed into the flask followed by the addition of(2R)-2-{[2-amino-5-(benzylthio)[1,3]thiazolo[4,5-d]pyrimidin-7-yl](methyl)amino}pentan-1-ol(5.43 g, 13.9 mmol). The resulting mixture was allowed to warm to −33°C. and sodium metal was added in small pieces until a blue colourappeared and persisted for 30 seconds. The reaction was then quenched byaddition of a spoon of solid ammonium chloride. The ammonia wasevaporated off and water (250 mL) was added to the residue. Theresulting mixture was neutralized with 1M hydrochloric acid (aq.). Theprecipitated product was collected by filtration, washed with water andacetonitrile and dried in vacuo to yield 3.38 g (81% yield) of the titlecompound.

¹H NMR (DMSO-d₆) 12.81 (br s, 1H); 8.45 (br s, 2H), 4.84 (br s, 1H),3.55-3.40 (m, 2H), 3.02 (s, 3H), 1.48 (m, 2H), 1.21 (m, 2H), 0.87 (t,3H); MS (ESI) m/z 300 [M+1]⁺.

e)(2R,2′R)-2,2′-{Dithiobis[(2-chloro[1,3]thiazolo[4,5-d]pyrimidine-5,7-diyl)(methylimino)]}dipentan-1-ol

(2R)-2-[(2-Amino-5-mercapto[1,3]thiazolo[4,5-d]pyrimidin-7-yl)(methyl)amino]pentan-1-ol(1.0 g, 3.34 mmol) was dissolved in acetonitrile (25 ml) andconcentrated hydrochloric acid (40 mL). Sodium nitrite (461 mg, 6.67mmol) was dissolved in water (2 mL) and added at 0° C. The reactionmixture was kept at 0° C. for three days. The reaction mixture waspoured onto ice and the precipitated product was collected by filtrationand washed with water. Drying in vacuo gave the title compound 800 mg(75% yield). MS (ESI) m/z 635 and 637 [M+1]⁺.

f)(2R,2′R)-2,2′-{Dithiobis[(2-methoxy[1,3]thiazolo[4,5-d]pyrimidine-5,7-diyl)(methylimino)]}dipentan-1-ol

Potassium hydroxide (210 mg, 3.75 mmol) dissolved in methanol (20 mL)was added to(2R,2′R)-2,2′-{dithiobis[(2-chloro[1,3]thiazolo[4,5-d]pyrimidine-5,7-diyl)(methylimino)]}dipentan-1-ol(795 mg, 1.25 mmol) in methanol (40 mL) at 0° C. The reaction wasstirred at 0° C. overnight and then the methanol was evaporated off. Theresidue was poured into ice and the resulting precipitate was collectedby filtration. The filtrate was extracted with ethyl acetate. Theorganic phase was dried over sodium sulphate and concentrated in vacuoand the residue was combined with the earlier collected solid to givethe title compound that was used in the next step without any furtherpurification. MS (ESI) m/z 627 [M+1]⁺.

g)5-[7-{[(1R)-1-(Hydroxymethyl)](methyl)amino}-[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one-5-yldisulfanyl]-7-{[(1R)-1-(hydroxymethylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

Crude(2R,2′R)-2,2′-{dithiobis[(2-methoxy[1,3]thiazolo[4,5-d]pyrimidine-5,7-diyl)(methylimino)]}dipentan-1-ol(1.25 mmol) from the previous step was dissolved in 1,4-dioxane (25 mL).Conc. hydrochloric acid (0.5 mL) and water (0.5 mL) was added and theresulting mixture was stirred at 45° C. over night. Dioxane wasevaporated in vacuo and the residue was poured onto ice to precipitatethe product that was collected by filtration. Drying in vacuo gave 590mg (78% yield over two steps) of the title compound. MS (ESI) m/z 599[M+1]⁺.

h)5-{[(1S)-1-(6-Chloropyridin-3-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)butyl](methyl)amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

The title compound was prepared in accordance with General method Gusing5-[7-{[(1R)-1-(hydroxymethyl)](methyl)amino}-[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one-5-yldisulfanyl]-7-{[(1R)-1-(hydroxymethylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one(0.10 g, 0.167 mmol), 2-chloro-5-[(1R)-1-chloroethyl]pyridine (Example6g, 0.065 g, 0.37 mmol) and sodium borohydride (0.038 g, 1.00 mmol),affording 0.060 g (41% yield) of the title compound.

¹H NMR (CDCl₃) δ ppm 8.56-8.38 (m, 1H), 7.87-7.73 (m, 1H), 7.28-7.26 (m,1H), 4.86 (q, 1H), 4.75-4.62 (m, 1H), 3.76-3.55 (m, 3H), 3.03 (s, 3H),1.70-1.63 (m, 3H), 1.53-1.45 (m, 2H), 1.26-1.21 (m, 2H), 0.95-0.88 (m,3H); MS (ESI) m/z 440 and 442 [M+1]⁺.

EXAMPLE 8 EXAMPLE 8a5-{[(1R)-1-(3-Fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-oneand EXAMPLE 8b5-{[(1S)-1-(3-Fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one

The diastereomeric mixture of5-{[1-(3-fluoropyridin-4-yl)ethyl]thio}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one(179 mg) from Example 3 was separated by preparative HPLC to yield 25 mgof the first eluting isomer:

¹H NMR (DMSO-d₆) δ ppm 12.31 (br s, 1H), 8.51 (m, 1H), 8.38 (d, 1H);7.62 (m, 1H); 6.97 (br s, 1H); 5.16 (q, 1H); 4.66 (t, 1H); 4.12 (m, 1H);3.44-3.30 (m, 2H, obscured by water signal), 1.66 (d, 3H), 1.61-1.27 (m,3H), 0.84 (d, 3H), 0.74 (d, 3H); MS (ESI) m/z 424 [M+1]⁺.

and 45 mg of the last eluting isomer:

¹H NMR (DMSO-d₆) δ ppm 12.35 (br s, 1H), 8.52 (d, 1H), 8.38 (d, 1H);7.62 (dd, 1H); 7.12 (br s, 1H); 5.15 (q, 1H); 4.62 (t, 1H); 4.21 (m,1H); 3.35-3.15 (m, 2H, partly obscured by water signal), 1.65 (d, 3H),1.63-1.29 (m, 3H), 0.88 (d, 3H), 0.85 (d, 3H); MS (ESI) m/z 424 [M+1]⁺.

Pharmacological Screens

Materials

Recombinant human fractalkine (hCX₃CL1) and recombinant humaninterleukin-8 (IL-8 or hCXCL8) were purchased from PeproTech Inc., UK.Recombinant [¹²⁵I]-fractalkine (human) and [¹²⁵I] hIL-8 with thespecific activity of 2200 Ci/mmol, was purchased from NEN® Life ScienceProducts, Inc., UK. Fluo4-AM was purchased from Molecular Probes, US.All other chemicals were of analytical grade.

Cells

The complete human CX₃CR1 cDNA (GenBank accession number U20350) wasextracted from human brain mRNA (Superscript, Life Technologies) andligated into pCR-Blunt II TOPO vector (InVitrogen). The insertcorresponding hCX₃CR1 was isolated and further subcloned intopcDNA3.1zeo. Plasmid DNA was prepared using Plasmid Midi Kit (Qiagen).Using Superfect Transfection Reagent (Qiagen) according to themanufacturer's protocol the expression plasmid for hCX₃CR1 was thenintroduced into human embryonic kidney suspension (HEKS) 293 cell linecontaining a vector for stable expression of a chimeric G-proteinGα_(qi5). A stable clone was generated utilizing zeocin (500 μg/mL) andhygromycin (100 μg/mL) selection. For further applications the cellswere maintained in Dulbecco's modified Eagle's medium/Ham's nutrient mixF12 (DMEM/F12) containing pyridoxine and supplemented with 10% (v/v)fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/mlstreptomycin, 250 mg/mL zeocin and 100 μg/mL hygromycin.

Cells expressing human CXCR2 obtained from AstraZeneca Chamwood arecultured in EMEM containing Glutamax and supplemented with 10% FBS (fromPAA, Austria), 1% non-essential amino acids (NEAA), 100 U/mL penicillinand 100 μg/mL streptomycin (PEST) and 500 μg/mL geneticin/G418.

Membrane Preparation

Cells are grown at 37° C. and 5% CO₂ and harvested at 60-80% confluencein buffer containing 10 mM Tris-HCl pH 7.4, 5 mM EDTA, 0.1 mg/mLbacitracin. The cells are centrifuged at 300×g for 10 min and the pelletis resuspended in harvesting buffer (10 mM Tris-HCl, pH 7.4, 5 mMethylenediaminetetra-aceticacid (EDTA) and 0.1 mg/mL bacitracin), pooledand homogenised using a Dounce homogeniser. The homogenate iscentrifuged in 48000×g for 10 min and resuspended in harvesting bufferusing Ultra-Turrax T8. Membrane aliquots are stored at −80° C. Proteinconcentration was determined in microtiter plates as described byHarrington (1990, Anal. Biochem. 186, 285-287).

In vitro Receptor Binding Assay

Competition binding studies of [¹²⁵I]fraktalkine were performed in 2 mL96-deep-well plates (Beckman, Germany) in a total volume of 1000μL/well. Each well contained 10 pM [¹²⁵I]-fractalkine and membraneequivalent to receptor concentration of 1 pM in assay buffer (50 mMHepes-KOH, pH 7.4, 10 mM MgCl₂, 1 mM EDTA, 0.1% (w/v) gelatine). Tenconcentrations (2 points/log unit) of the test compounds werepre-dissolved in DMSO and added to reach a final concentration of 1%(v/v) DMSO. The assay was initiated with the addition of membranes andincubated at 25° C. for 24 h. The reactions were stopped by rapidfiltration through Whatman GF/B glass fiber filters pretreated with 0.3%polyethylimine and subsequent washing with ice-cold buffer (10 mMHepes-KOH pH 7.4, 500 mM NaCl) using a Brandel receptor bindingharvester. Scintillation cocktail was added and radioactivity wasdetermined in a Packard 2500TR liquid scintillation counter. (PerkinElmer, USA)

The [¹²⁵I]-hIL-8 competition binding studies are performed insinglicates in white clear bottom 96-well isoplates with a final volumeof 200 μL and each well contains 150 pM [¹²⁵I]-hIL-8 (specific activity2200 Ci/mmol), membrane-SPA preparation equivalent to 20 pM receptorsand 1.5 mg SPA-beads in assay buffer [50 mM HEPES-KOH pH 7.4, 10 mMMgCl₂, 1 mM EDTA, 0.5% (w/v) gelatin]. The test compounds were treatedas above. The non-specific binding is determined in the presence of 500nM unlabelled hIL-8 The agonist hIL-8 (a concentration-response curvefrom 3 pM to 30 nM), is used as reference compound at each testoccasion. The peptide curve does not contain DMSO. The binding reactionis started by addition of 140 μL membrane-SPA preparation, and thesamples are incubated in dark at RT for 4 h. Assay plates are counted ina liquid scintillation counter (Wallac MicroBeta® TriLux 1450 fromPerkinElmer, USA).

[³⁵S]GTPγS Binding

The [³⁵S]GTPγS binding studies were carried out in clear-bottommicrotiter plates in duplicates with 10 concentrations of the inhibitor(2 conc/log units) diluted in DMSO (final conc 1%) and at roomtemperature. Membranes expressing the hCX₃CR1 receptor (finalconcentration 20 μg protein/well) were added together with SPA beads(final concentration 1 mg/well) all suspended in GTPγS binding buffer(50 mM Tris-HCl, 100 mM NaCl, 0.1% gelatin, 15 μg saponin/mL and 3 μMGDP, pH 7.4 at rt). Membranes, SPA beads and drugs were pre-incubated 30min before addition of 310 pM fraktalkine for maximal stimulation. Basalactivity was defined as the activity found without fraktalkinestimulation (GTPγS binding buffer). After additional 30 min the reactionwas started with the addition of [³⁵S]GTPγS to a final concentration of0.1 nM and a final assay volume of 0.2 mL. The experiment was terminated30 minutes later by centrifugation at 2000 rpm for 2×5 minutes(different directions) and the radioactivity determined in a liquidscintillation counter (Wallac MicroBeta® TriLux 1450).

Results

Typical CX₃CR1 Ki values for the compounds of the present invention arein the range of about 0.1 to about 1000 nM. Other values for CX₃CR1 Kiare in the range of about 0.1 nM to about 500 nM. Further values forCX₃CR1 Ki are in the range of about 0.1 nM to about 25 nM. Results fromin vitro hCX₃CR1 binding assay for final compounds are shown in Table 1.

TABLE 1 Example no K_(i) (nM) 1 5.8 2 20 3 Not tested* 4 18 5 Nottested** 6 21.4 7 440 8a 97 8b 1.5 *diastereomeric mixture of examples8a and 8b. **not available in enough quantity for testing in the invitro hCX₃CR1 binding assay.

The compounds of the present invention wherein R¹ represents Me(containing a branched thioalkylpyridyl group in 5-position) are bothmore potent antagonists at the CX₃CR1 receptor and/or less potentantagonists at the CXCR2 receptor than corresponding reference compoundswherein R¹ represents H. Such enhanced selectivity with respect toantagonism of the CX₃CR1 receptor is expected to result in significanttherapeutic benefit.

1-20. (canceled)
 21. A method of treating, or reducing the risk ofneurodegenerative disorders, demyelinating disease, cardio- andcerebrovascular atherosclerotic disorders, peripheral artery disease,rheumatoid arthritis, pulmonary diseases such as COPD, asthma or pain,comprising administering to a person suffering from or susceptible tosuch a disease or condition, a therapeutically effective amount of acompound of formula (I),

wherein: R¹ is CH₃ or CF₃; R² is halo, CN or C₁₋₆alkyl; R³ is H or CH₃;R⁴ is H or CH₃; and n is 0, 1 or 2, or a pharmaceutically acceptablesalt thereof.
 22. A method of treating, or reducing the risk of multiplesclerosis, comprising administering to a person suffering from orsusceptible to such a disease or condition, a therapeutically effectiveamount of a compound of formula (I),

wherein: R¹ is CH₃ or CF₃; R² is halo, CN or C₁₋₆alkyl; R³ is H or CH₃;R⁴ is H or CH₃; and n is 0, 1 or 2, or a pharmaceutically acceptablesalt thereof.
 23. A process for the preparation of a compound of formula(I), as defined in claim 21, or a pharmaceutically acceptable saltthereof, comprising: a) reacting a compound of formula (II):

wherein R³ and R⁴ are as defined in formula (I); with a compound offormula (III):

wherein R¹, R² and n are as defined in formula (I) and L¹ represents aleaving group; and where necessary converting the resultant compound offormula (I), or another salt thereof, into a pharmaceutically acceptablesalt thereof; or converting the resultant compound of formula (I) into afurther compound of formula (I); and where desired converting theresultant compound of formula (I) into an optical isomer thereof.
 24. Aprocess for the preparation of a compound of formula (I), as defined inclaim 21, or a pharmaceutically acceptable salt thereof, comprising: a)hydrolysing a compound of formula (IV)

wherein R¹, R², R³, R⁴ and n are as defined in formula (I); and wherenecessary converting the resultant compound of formula (I), or anothersalt thereof, into a pharmaceutically acceptable salt thereof; orconverting the resultant compound of formula (I) into a further compoundof formula (I); and where desired converting the resultant compound offormula (I) into an optical isomer thereof.
 25. A method of treating, orreducing the risk of multiple sclerosis, comprising administering to aperson suffering from or susceptible to such a disease or condition, apharmaceutical composition comprising a compound of formula (I), asdefined in claim 22, as a free base or a pharmaceutically acceptablesalt thereof, in admixture with a pharmaceutically acceptable diluent orcarrier.
 26. A method of treating, or reducing the risk of multiplesclerosis, comprising administering to a person suffering from orsusceptible to such a disease or condition, a therapeutically effectiveamount of a compound that is5-{[(1S)-1-(5-chloropyridin-2-yl)ethyl]thio}-7-{[(1R)-1(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one,or a pharmaceutically acceptable salt thereof.
 27. A method of treating,or reducing the risk of multiple sclerosis, comprising administering toa person suffering from or susceptible to such a disease or condition, apharmaceutical composition comprising a compound according to claim 26,or a pharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable diluent or carrier.